Supplementary Figure 6: Inactivation of FHOD4, similar to IRAP deficiency, increases the activation of TLR9.
From: IRAP+ endosomes restrict TLR9 activation and signaling
(a) WT and KO BMDCs were transduced with shNT (non-targeting) and shFHOD4 (17) lentiviruses and stimulated with different TLR ligands for 6 h. The secretion of IL-12p40 and IL-6 in supernatants was measured by ELISA (n=2 experiments, mean ± SEM, **p<0.009, *p<0.018). (b) BMDCs from TLR9-GFP transgenic mice were transduced with lentiviruses coding for 5 different shRNA against FHOD1 (shFHOD1 42, 91, 47, 49, 02) or a non-targeting shRNA (shNT) and the knock-down efficiency was analyzed by immunoblotting with antibodies specific for FHOD1. WT fibroblasts transfected with a plasmid coding for FHOD1 were used as positive control for anti-FHOD1 antibodies. (c-d) BMDCs from TLR9-GFP transgenic mice were transduced with lentiviruses expressing shNT or shFHOD1 (42) and used to analyze TLR9-GFP localization in steady state conditions by confocal microscopy using an anti-LAMP1 antibody (c) or to measure the secretion of IL-6 and IL-12(p40) after TLR4 and TLR9 activation (d) (n=2 experiments, mean ± SEM).
